title: Production of a recombinant antibody specific for i blood group antigen, a mesenchymal stem cell marker.
abstract: Multipotent mesenchymal stem/stromal cells (MSCs) offer great promise for future regenerative and anti-inflammatory therapies. Panels of functional and phenotypical markers are currently used in characterization of different therapeutic stem cell populations from various sources. The i antigen (linear poly-N-acetyllactosamine) from the Ii blood group system has been suggested as a marker for MSCs derived from umbilical cord blood (UCB). However, there are currently no commercially available antibodies recognizing the i antigen. In the present study, we describe the use of antibody phage display technology to produce recombinant antibodies recognizing a structure from the surface of mesenchymal stem cells. We constructed IgM phage display libraries from the lymphocytes of a donor with an elevated serum anti-i titer. Antibody phage display technology is not dependent on immunization and thus allows the generation of antibodies against poorly immunogenic molecules, such as carbohydrates. Agglutination assays utilizing i antigen-positive red blood cells (RBCs) from UCB revealed six promising single-chain variable fragment (scFv) antibodies, three of which recognized epitopes from the surface of UCB-MSCs in flow cytometric assays. The amino acid sequence of the VH gene segment of B12.2 scFv was highly similar to the VH4.21 gene segment required to encode anti-i specificities. Further characterization of binding properties revealed that the binding of B12.2 hyperphage was inhibited by soluble linear lactosamine oligosaccharide. Based on these findings, we suggest that the B12.2 scFv we have generated is a prominent anti-i antibody that recognizes i antigen on the surface of both UCB-MSCs and RBCs. This binder can thus be utilized in UCB-MSC detection and isolation as well as in blood group serology.
authors.affiliation: Finnish Red Cross Blood Service , Helsinki, Finland .
authors.forename: Tia
authors.initials: T
authors.lastname: Hirvonen
authors.forename: Heli
authors.initials: H
authors.lastname: Suila
authors.forename: Sari
authors.initials: S
authors.lastname: Tiitinen
authors.forename: Suvi
authors.initials: S
authors.lastname: Natunen
authors.forename: Marja-Leena
authors.initials: ML
authors.lastname: Laukkanen
authors.forename: Annika
authors.initials: A
authors.lastname: Kotovuori
authors.forename: Mirka
authors.initials: M
authors.lastname: Reinman
authors.forename: Tero
authors.initials: T
authors.lastname: Satomaa
authors.forename: Kaija
authors.initials: K
authors.lastname: Alfthan
authors.forename: Saara
authors.initials: S
authors.lastname: Laitinen
authors.forename: Kristiina
authors.initials: K
authors.lastname: Takkinen
authors.forename: Jarkko
authors.initials: J
authors.lastname: Räbinä
authors.forename: Leena
authors.initials: L
authors.lastname: Valmu
keywords: MSC; i blood group antigen; phage display; recombinant antibody development